幽门螺杆菌Catalase基因的克隆、表达及其抗原性鉴定

目的：构建含人幽门螺杆菌（Hpylori，Hp）过氧化氢酶（catalase，KatA）编码基因的重组质粒，测定、分析其核酸序列，并在E.coli中表达，研究其抗原性。方法：应用PCR技术从HpDNA染色体中扩增KatA编码基因片段，将其T-A克隆和测序，并与GenBank公布的其他Hp菌株的基因序列比较，再将目的基因插入至融合表达载体pGEX-4T-1中进行表达，用GST亲和层析对其进行纯化。纯化产物用于对29株小鼠抗Hp-全菌单克隆抗体（mAb）的鉴定及与Hp啊感染患者血清进行Western blot。结果：KatA基因全长为1 515 bp，并在GenBank上登录（No．DQ333889），与GenBank公布的其他Hp菌株的核酸的同源性为96％～97％，表达的KatA融合蛋白的相对分子质量（Mr）为85 000，29株小鼠抗Hp全菌mAb中有4株mAb是针对KatA的，表达产物可被Hp感染患者的血清特异性识别。结论：重组KatA具有较好的抗原性，为坳检测试剂和疫苗的研究奠定了基础。

Cloning, expression and identification of catalase of Helicobacter pylori

AIM: To construct the recombinant plasmid containing catalase(KatA) of Helicobacter pylori(Hp), analyze its nucleic acid sequence, express it in E.coli and study its antigenicity. METHODS: KatA fragments were amplified from Hp chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other HP strains on the GenBank. Then the gene cloned into pGEX-4T-1 fusion expression vector was expressed in E.coli and purified by GST-affinity chromatography. The purified product was used to identify 29 stains of mouse anti Hp monoclonal antibodies and analyze antigenicity with serum of Hp-infected patients by Western blot. RESULTS: KatA fragments were composed of 1 515 bp (GenBank No.DQ333889) and the nucleotide homology with other Hp strains on the GenBank was 96%-97% . 85 kDa of the recombinant KatA-pGEX-4T-1 was expressed in E.coli. 4 of 29 anti-Hp mouse monoclonal antibodies were against KatA. Western blot analysis proved that KatA was specifically recognized in the serum of Hp-infected patients. CONCLUSION: The recombinant KatA has original antigenicity. It is of great value to clinical sero-diagnosis and vaccine study of Hp.